Digital recordings of gas-vesicle collapse used to measure turgor pressure and cell-water relations of cyanobacterial cells.

The gas vesicles of the cyanobacterium Microcystis sp. collapse under pressures ranging from 0.65-1.10 MPa, determined from turbidity changes in a pressure nephelometer. In turgid cells, collapse occurs at a lower range of pressures; the difference is equal to the cell turgor pressure. The turgor pressure decreases, however, as gas vesicles collapse; this decrease is minimised by calculating the turgor pressure in samples with few of their gas vesicles collapsed. Previously, pressure and turbidity were measured in discrete steps, using analogue meters, or continuously, using chart recorders: turgor pressure was calculated from the mean or median collapse pressures. We describe modifications allowing continuous digital recording; the output was modelled with polynomial or sigmoid functions, the latter providing the best fit over the full collapse-pressure curve; turgor pressure could then be calculated for any point on the collapse-pressure curve. The shape of the collapse-pressure curve was affected by the rate of pressure rise; curves were similar to those from step-wise methods when the pressure was raised at approximately 4 kPa s(-1). Under a rapid, almost instantaneous, rise in pressure there was a larger initial decrease in turgor and from the subsequent recovery the hydraulic conductivity of the cell surface could be calculated; the new method gave improved measurements of the cell volumetric elastic modulus. Following collapse of half the gas vesicles, cells recovered their full turgor pressure after 3 h. This suggests turgor homeostasis. These methods are applicable to other bacteria with gas vesicles, including Escherichia coli, if it could be genetically modified to express transgenic gas vesicles.

Changes in cell turgor pressure related to uptake of solutes by Microcystis sp. strain 8401.

Uptake of several naturally occurring organic solutes by the unicellular cyanobacterium Microcystis sp. caused changes in cell turgor pressure (p(t)), which was determined by measuring the mean critical pressure (p(c)) of gas vesicles in the cells. Cells had an initial p(t) of 0.34 MPa, which decreased to 0.08 MPa in 0.15 M sucrose. In solutions of polyols, p(t) gradually recovered as the solutes penetrated the cytoplasmic membranes. From measurements of the exponential rate of turgor increase, cell volume and surface area, the permeability coefficient of the cytoplasmic membrane to each solute was calculated. Permeabilities to amino acids, ammonium ions and sodium acetate indicated little passive movement of these substances across the cell surface from solutions at high concentrations. We looked for evidence of ion trapping of acetic acid: at low pH there was a rapid rise in turgor pressure indicating a rapid uptake of this weak acid. After 20 min the turgor was lost, apparently due to loss of integrity of the cell membranes. For cells in natural habitats, studies of the permeability of cells to solutes is relevant to the problem of retaining substances that are accumulated by active uptake from solutions of low concentrations in natural waters.

Cyanobacterial heterocysts: terminal pores proposed as sites of gas exchange.

In many filamentous cyanobacteria, oxygenic photosynthesis is restricted to vegetative cells, whereas N(2) fixation is confined to microoxic heterocysts. The heterocyst has an envelope that provides a barrier to gas exchange: N(2) and O(2) diffuse into heterocysts at similar rates, which ensures that concentrations of N(2) are high enough to saturate N(2) fixation while respiration maintains O(2) at concentrations low enough to prevent nitrogenase inactivation. I propose that the main gas-diffusion pathway is through the terminal pores that connect heterocysts with vegetative cells. Transmembrane proteins would make the narrow pores permeable enough and they might provide a means of regulating the rate of gas exchange, increasing it by day, when N(2) fixation is most active, and decreasing it at night, minimizing O(2) entry. Comparisons are made with stomata, which regulate gas exchange in plants.

The uptake of amino acids by the cyanobacterium Planktothrix rubescens is stimulated by light at low irradiances.

The rates of uptake of five amino acids--alanine, glutamate, glycine, leucine and serine--by axenic cultures of the cyanobacterium Planktothrix rubescens were measured over a range of irradiances using the (14)C-labelled amino acids at the nanomolar concentrations observed in Lake Zürich. The rates in the light exceeded the dark rates by as much as two- to ninefold. The light-affinity constants for stimulation were similar, indicating a similar process for each of the five amino acids. The E(k) (light saturation irradiance) for light stimulation was only 1 micromol m(-2) s(-1), less than the compensation point for photosynthesis and autotrophic growth, and much lower than the E(k) for either process. The E(k) for amino acid uptake was also less than the irradiance at which filaments obtain neutral buoyancy, which determines the depth at which they stratify and the irradiance they receive. This indicates that stimulation of amino acid uptake by light of low irradiances provides a mechanism for supplementing growth of filaments stratifying deep in the metalimnion, which, while able to grow at low irradiances, are often left with insufficient light to sustain them. Acetate uptake was also stimulated by light, but the kinetics differed.

Sinking velocities of phytoplankton measured on a stable density gradient by laser scanning.

Two particular difficulties in measuring the sinking velocities of phytoplankton cells are preventing convection within the sedimenting medium and determining the changing depth of the cells. These problems are overcome by using a density-stabilized sedimentation column scanned by a laser. For freshwater species, a suspension of phytoplankton is layered over a vertical density gradient of Percoll solution; as the cells sink down the column their relative concentration is measured by the forward scattering of light from a laser beam that repeatedly scans up and down the column. The Percoll gradient stabilizes the column, preventing vertical mixing by convection, radiation or perturbation of density by the descending cells. Measurements were made on suspensions of 15 microm polystyrene microspheres with a density of 1050 kg m-3; the mean velocity was 6.28 microm s-1, within 1.5% of that calculated by the Stokes equation, 6.36 microms-1. Measurements made on the filamentous cyanobacterium Planktothrix rubescens gave mean velocities within the theoretical range of values based on the range of size, shape, orientation and density of the particles in a modified Stokes equation. Measurements on marine phytoplankton may require density gradients prepared with other substances.

Analysis of tryptic digests indicates regions of GvpC that bind to gas vesicles of Anabaena flos-aquae.

The gas vesicles of the cyanobacterium Anabaena flos-aquae contain two main proteins: GvpA, which forms the ribs of the hollow cylindrical shell, and GvpC, which occurs on the outer surface. Analysis by MALDI-TOF MS shows that after incubating Anabaena gas vesicles in trypsin, GvpA was cleaved only at sites near the N-terminus, whereas GvpC was cleaved at most of its potential tryptic sites. Many of the resulting tryptic peptides from GvpC remained attached to the underlying GvpA shell: the pattern of attachment indicated that there are binding sites to GvpA at both ends of the 33-residue repeats (33RRs) in GvpC, although one of the tryptic peptides within the 33RR did not remain attached. Tryptic peptides near the two ends of the GvpC molecule were also lost. The mean critical collapse pressure of Anabaena gas vesicles decreased from 0.63 MPa to 0.20 MPa when GvpC was removed with urea or fully digested with trypsin; partial digestion resulted in partial decrease in critical pressure.

Gas vesicles in actinomycetes?

Gas vesicles encoded by gvp genes provide buoyancy in many prokaryotes. In a recent Trends in Microbiology article entitled 'Gas vesicles in actinomycetes: old buoys in novel habitats?' van Keulen et al. documented the occurrence of gvp genes in soil-inhabiting actinomycetes but questioned whether any of them produce gas vesicles. We suggest that the protein encoded by gvpA in actinomycetes might be incompatible with the structure of the standard gas vesicle. Perhaps it has another role associated with the air-water interface.

The Burgundy-blood phenomenon: a model of buoyancy change explains autumnal waterblooms by Planktothrix rubescens in Lake Zürich.

Buoyancy changes of the cyanobacterium Planktothrix rubescens- the Burgundy-blood alga - were modelled from its buoyancy response to light and irradiance changes in Lake Zürich during autumnal mixing. The daily insolation received by filaments at fixed depths and circulating to different depths was calculated from the measured light attenuation and surface irradiance. The active mixing depth, za5, was determined from the vertical turbulent diffusion coefficient, Kz, calculated from the wind speed, heat flux and temperature gradients. The fixed depth resulting in filament buoyancy, zn, decreased from 13 to 2 m between August and December 1998; the critical depth for buoyancy, zq, to which filaments must be circulated to become buoyant, decreased from >60 m in the summer to <10 m in winter. When za5 first exceeded zn, in September, P. rubescens was mixed into the epilimnion. In October, zq > za5: circulating filaments would have lost buoyancy in the high insolation. Often in November and December, after deeper mixing and lower insolation, za5 > zq: filaments would have become buoyant but would have floated to the lake surface (the Burgundy-blood phenomenon) only under subsequent calm conditions, when Kz was low. The model explains the Burgundy-blood phenomenon in deeper lakes; waterblooms near shallow leeward shores arise from populations floating up in deeper regions of the lake.

Stratification by cyanobacteria in lakes: a dynamic buoyancy model indicates size limitations met by Planktothrix rubescens filaments.

The ability of the Planktothrix rubescens to stratify in Lake Zürich is related to the size and shape of the cyanobacterial filaments. Detailed measurements made in the lake are used in a dynamic computer model of buoyancy regulation to investigate the vertical movements of filaments tracking the depth at which the irradiance would support neutral buoyancy. The movement of the filament lags behind the constantly changing target depth owing to (a) the time taken for the filament to respond to the irradiance by changing its density and (b) the time it takes to move by sinking down or floating up through the water column. The model simulates the stratification depth over a 5-month period of the summer from the continuous measurements of irradiance and weekly measurements of light attenuation and temperature, without any further adjustment over the period. Models using filaments of the size observed in Lake Zürich explain several details of the observed depth changes: smaller planktonic cyanobacteria (e.g. Limnothrix sp.) are unable to migrate fast enough and larger ones (e.g. Anabaena spp.) will overshoot and become entrained in the epilimnion. The model can be used to simulate recruitment of Planktothrix filaments from different depths after vernal stratification. Recruitment of filaments from depths down to 45 m will contribute to the metalimnetic population increase in early July.

The diameter and critical collapse pressure of gas vesicles in Microcystis are correlated with GvpCs of different length.

In cyanobacteria the protein on the outside of the gas vesicle, GvpC, is characterised by the presence of a 33 amino acid residue repeat (33RR), which in some genera is highly conserved. The number of 33RRs correlates with the diameter of the gas vesicle and inversely with its strength. Gas vesicles isolated from Microcystis aeruginosa strain PCC 7806 were found to be wider and have a lower critical collapse pressure than those from Microcystis sp. strain BC 8401. The entire gas-vesicle gene cluster of the latter strain was sequenced and compared with the published sequence of the former: the sequences of nine of the ten gvp genes differed by only 1-5% between the two strains; the only substantial difference was in gvpC which in strain BC 8401 lacked a 99-nucleotide section encoding a 33RR. This observation further narrows the correlation of gas vesicle width to the number of 33RRs and suggests how Microcystis strains might be used in experimental manipulation of gas vesicle width and strength.

Archaea with square cells.

Two groups of microbiologists have independently isolated 'Walsby's square bacterium' from salt crystallizer ponds; its growth depends on pyruvate. Genetic analysis shows that the squares, discovered 25 years ago on the Sinai Peninsula, are archaea rather than bacteria. These transparent tile-like cells might have been dismissed as surface artefacts of salt crystals but for their gas vesicles--structures peculiar to prokaryotic organisms. Paradoxically, the square archaea are the dominant microorganisms in some hypersaline environments and might be globally important.

Different gvpC length variants are transcribed within single filaments of the cyanobacterium Planktothrix rubescens.

Transcripts of the gas vesicle genes gvpA and gvpC were detected in single filaments of the cyanobacterium Planktothrix rubescens using reverse transcription and quantitative real-time PCR. Primers were designed to amplify short sequences within gvpA and three length variants of gvpC. With genomic template DNA, and using Sybr Green to monitor product accumulation, similar amplification efficiencies were observed for each of these genes. The relative copy numbers of gvpC length variants in genomic DNA from five Planktothrix gas vesicle genotypes determined by real-time PCR were similar to those indicated by sequencing the gas vesicle gene clusters. The precipitation of gvp cDNA reverse-transcribed from cellular RNA from single filaments was required before amplification of the gene fragments; without this step it was not possible to detect the accumulation of the expected amplicons by dissociation analysis. Precipitation was also necessary to ensure the generation of product curves that allowed linear regression in an early stage of PCR, a prerequisite for the quantification of low-input cDNA amounts without the need for standard curves. This report shows that different gvpC length variants are transcribed within single Planktothrix filaments, both from laboratory cultures and from natural samples taken from Lake Zurich. This has implications for the efficiency of buoyancy provision by the possible production of gas vesicles of different strengths within individual cyanobacterial filaments. The hypothesis that post-transcriptional regulation may influence the type of protein (GvpC) present in gas vesicles is presented.

Spontaneous mutations in gas vesicle genes of Planktothrix spp. affect gas vesicle production and critical pressure.

Wild-type strains of the cyanobacterium Planktothrix rubescens have a cluster of gas vesicle (gvp) genes with repeats of alternating gvpA and gvpC. The gvpC occurs in three length variants, all with the same 3'-sequence, OmegaC. Spontaneous non-buoyant mutants had lost some of the alternating gvpAC copies and their gvpC genes had a novel 3'-end sequence, PsiC; additional gvpC genes terminating in this sequence were also found in the wild-type and representatives of other GV genotypes. Alleles of gvpC terminating in PsiC occurred only at the downstream ends of the gvpAC clusters investigated; all other gvpCs terminated in OmegaC. Mutants of strains with the GV3 genotype produced only 30-50% of the gas vesicles present in the wild-type; their gas vesicles had lower mean critical pressures (0.70-0.78 MPa) than those in the wild-type (1.05-1.10 MPa).

The sequence of the major gas vesicle protein, GvpA, influences the width and strength of halobacterial gas vesicles.

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.

The daily integral of growth by Planktothrix rubescens calculated from growth rate in culture and irradiance in Lake Zürich.

In Lake Zürich, populations of the cyanobacterium Planktothrix rubescens develop in the metalimnion during the summer and become gradually entrained in the deepening surface mixed layer during the autumn. It had previously been demonstrated that the daily integrals of photosynthetic production accounted for the growth observed in the metalimnion and greatly exceeded the smaller increase during the autumn. We have now determined the relationship between growth rate (µ) and irradiance (I) in cultures of P. rubescens strain Pla 9316 maintained at 20 °C on a 12∶12 h light∶dark cycle: the highest net growth rate averaged over the 24 h (µh ) was 0.123 d-1 , the dark rate (µD ) was -0.020 d-1 , the gross rate (φm = µh -µD ) was 0.144 d-1 , the affinity coefficient (α) was 0.0273 (d µmol m-2 s-1 )-1 and the compensation point (IC ) was 1.76 µmol m-2 s-1 . Using the corresponding coefficients calculated for the light period (µLh = 0.267 d-1 , φLm = 0.287 d-1 and αL = 0.0547 (d µmol m-2 s-1 )-1 ), instantaneous growth rates could be calculated from the irradiance. Comparison with growth rates at 10 °C indicated a Q10 of 1.48. These coefficients were used in a modification of the Smith equation to calculate potential growth rates of Planktothrix from the irradiance and temperature at each time and depth in Lake Zürich. Data on irradiance, vertical light attenuation and temperature were used to calculate the daily integrals of biomass increase over a period of 136 d. These growth integrals gave a closer correspondence to the observed population increase than the photosynthetic integrals calculated previously from measurements made with lakewater samples dominated by Planktothrix. Photosynthetic measurements made with the Planktothrix culture indicated a maximum rate of carbon increase (0.467 d-1 that exceeds the maximum growth rate, which suggests that other factors limit growth over long periods.

Growth competition between Halobacterium salinarium strain PHH1 and mutants affected in gas vesicle synthesis.

To investigate the role of the buoyancy provided by gas vesicles in the facultative anaerobe Halobacterium salinarium PHH1, the growth of a gas-vacuolate (Gv+) strain in competition with two gas-vesicle-defective (Gvdef) mutants was examined. The Gv+ strain synthesized gas vesicles throughout its growth cycle, and floated up to form a thick surface scum during the exponential growth phase in static culture. Mutant Gvdef1 produced significantly fewer gas vesicles than the Gv+ strain in corresponding stages of growth, although in late stationary phase a small proportion of cells floated up to the surface of static cultures. Mutant Gvdef2 had a much lower gas vesicle content in shaken culture and produced negligible amounts of gas vesicles in static culture. The Gv+ and the two Gvdef strains grew equally well in shaken cultures, but in static cultures, where steep vertical gradients of oxygen concentration were established, Gvdef1 was outgrown by the Gv+ strain. Gvdef2 outcompeted the Gv+ strain in shallow static cultures, perhaps because Gvdef2 carried a smaller protein burden, which offset the benefits of buoyancy. This selection for Gvdef2 was lost in deeper static cultures, although it could be restored by aerating static cultures from below. The results support the hypothesis that the role of buoyancy in halobacteria is to maintain cells at the more aerated surface of brine pools.

The selective advantage of buoyancy provided by gas vesicles for planktonic cyanobacteria in the Baltic Sea.

Observations were made on the vertical distribution of colonies of Aphanizomenon flos-aquae for 9 d at a drift-station east of Bornholm Island in the Baltic Sea. The buoyant colonies were dispersed in the upper layers of the water column during periods of wind-induced mixing but floated up during calm periods. From measurements of the vertical light extinction, surface irradiance and the photosynthesis versus irradiance curve, calculations were made of the changes in the daily integral of photosynthesis with respect to time and depth throughout the water column. From these calculations it is demonstrated that net photosynthesis by the population of Aphanizomenon flos-aquae increased nearly threefold by floating up after a deep mixing event. It is estimated that, averaged over alternating periods of calm and mixing, the buoyancy provided by gas vesicles in this organism will result in a nearly twofold increase in photosynthesis. A quantitative analysis has been made of the relationship of the daily integral of photosynthesis by the Aphanizomenon population with the mean depth of the population in the water column and the daily insolation. The analysis shows that the integral decreases linearly with respect to mean depth.